Helicobacter pylori Antimicrobial Resistance in a Pediatric Population From the New England Region of the United States
Authors: Michael Herzlinger, Katelyn Dannheim, Muhammad Riaz, Enju Liu, Athos Bousvaros, Silvana Bonilla
Journal: Clinical Gastroenterology and Hepatology (2023)

Helicobacter pylori (H pylori) infection remains common worldwide, with an estimated overall prevalence of 33% in pediatric population.1Yuan C. et al.Lancet Child Adolesc Health. 2022; 6: 185-194Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Growing data demonstrate a global rise in the prevalence of H pylori antimicrobial resistance.2Savoldi A. et al.Gastroenterology. 2018; 155: 1372-1382.e17Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar Pediatric gastroenterology guidelines strongly recommend obtaining H pylori antimicrobial susceptibility before prescribing treatment when possible.3Jones N. et al.J Pediatr Gastroenterol Nutr. 2017; 64: 991-1003Crossref PubMed Scopus (284) Google Scholar Because of several reasons, including limited availability of gastric biopsy culture, U.S. antimicrobial susceptibility data are limited.4Bhakta D. et al.Clin Gastroenterol Hepatol. 2018; 16: 1531-1532Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar,5Bonilla S. et al.JPGN Reports. 2021; 2: e116Crossref Google Scholar Clarithromycin and levofloxacin resistance rates of 15%–30% have been reported from single-center small adult studies.6Duck W. et al.Emerg Infect Dis. 2004; 10: 1088-1094Crossref PubMed Scopus (179) Google Scholar, 7Mosites E. et al.J Glob Antimicrob Resist. 2018; 15: 148-153Crossref PubMed Scopus (21) Google Scholar, 8Argueta E.A. et al.Gastroenterology. 2021; 160: 2181-2183.e1Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar However, data in children are lacking. We determined the rate of H pylori antimicrobial resistance in formalin-fixed paraffin embedded gastric tissue from pediatric patients from 2 New England tertiary care centers using a novel next generation sequencing (NGS)-based molecular method with proven reliability in adult population.9Hulten K. et al.Gastroenterology. 2021; 161: 1433-1442.e2Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar We compared results with gastric biopsy culture antimicrobial susceptibilities via agar dilution in a subset of patients and evaluated the impact of antimicrobial resistance on eradication rates of empiric therapies. Electronic medical records of patients with H pylori diagnostic codes evaluated at Boston Children's Hospital, Boston, Massachusetts and Hasbro Children's Hospital, Providence, Rhode Island between January 2015 and September 2019 were reviewed. We included patients with H pylori identified on gastric biopsy who had tissue blocks available for analysis. Demographic variables, first H pylori treatment, and eradication results were collected. H pylori antimicrobial resistance profiles were obtained via NGS-based molecular method using PyloriAR (American Molecular Laboratories, Vernon Hills, IL). Off-site specialty laboratory performed H pylori gastric biopsy culture (Supplementary Methods). We included 274 patients (102 from Boston,172 from Providence). Mean age was 12.7 ± 4.6 years; 151 (55%) were female, 164 (60%) were white, and 95 (35%) were Hispanic. Twenty-three cases (23/274, 8.4%) were excluded from the analysis because of insufficient H pylori DNA. One hundred nine cases (109/251, 43.4%) were resistant to at least 1 antimicrobial. Clarithromycin and metronidazole were the antimicrobials with the highest resistance rates, 23.5% and 22%, respectively. Treatment data were available in all 235 treated patients. The most common treatment regimens included proton pump inhibitor (PPI)-clarithromycin-amoxicillin (53.2%), PPI-metronidazole-amoxicillin (23%), and PPI-tetracycline-metronidazole-bismuth (6%). Pediatric societal recommendations guided dosing and duration of treatment.3Jones N. et al.J Pediatr Gastroenterol Nutr. 2017; 64: 991-1003Crossref PubMed Scopus (284) Google Scholar Eradication data were available for 61.3% of treated patients (144/235). The overall eradication rate was 73.6% (106/144). Quadruple therapies had the highest eradication rates (≥90%). Eradication rates for triple therapies were less than 75%. Table 1 shows antimicrobial resistance rates, treatment regimens, and eradication rates. Resistance profiles by treatment are provided in Supplementary Table 1.Table 1Antimicrobial Resistance, Treatment Regimens, and Eradication RatesAntimicrobial (n = 251)Resistance, % (n) Resistance to ≥1 antibiotics43.4 (109)Clarithromycin23.5 (59)Metronidazole21.9 (55)Fluoroquinolones8.4 (21)Rifabutin3.6 (9)Amoxicillin2.4 (6)Tetracycline<1 (1)Co-resistance patterns (n = 29 isolates resistant to >1 antimicrobials) Clarithromycin-metronidazolen = 10 Clarithromycin-rifabutinn = 1 Clarithromycin-amoxicillinn = 1 Clarithromycin-tetracyclinen = 1 Metronidazole-fluoroquinolonesn = 4 Metronidazole- rifabutinn = 2 Clarithromycin-metronidazole-fluoroquinolonesn = 4 Clarithromycin-amoxicillin-fluoroquinolonesn = 2 Metronidazole-fluoroquinolones-rifabutinn = 1 Clarithromycin-metronidazole-amoxicillin-fluoroquinolonen = 2 Clarithromycin-metronidazole-fluoroquinolone-rifabutinn = 1Treatment regimen (total [%])Eradication success (%)All treatment regimens (144 [100])aDual regimen PPI-amoxicillin (1 [0.7], 100% eradication). Triple regimensPPI-clarithromycin-amoxicillin (78 [54.2])58 (74.4)PPI-metronidazole-amoxicillin (29 [20.1])19 (65.5)PPI-clarithromycin-metronidazole (5 [3.5])3 (60.0)PPI-clarithromycin-amoxicillin clavulanate (1 [0.7])1 (100.0)PPI-amoxicillin-doxycycline (1 [0.7])1 (100.0)PPI-metronidazole-bismuth (1 [0.7])0 (0) Quadruple regimensPPI-amoxicillin-metronidazole-bismuth (8 [5.6])4 (50.0)PPI-tetracycline-metronidazole-bismuth (11 [7.6])10 (90.9)PPI-doxycycline-metronidazole-bismuth (1 [0.7])1 (100.0)PPI-clarithromycin-amoxicillin-metronidazole (5 [3.5])5 (100.0)PPI-clarithromycin-amoxicillin-bismuth (1 [0.7])1 (100.0)PPI-clarithromycin-metronidazole-tetracycline (1 [0.7])1 (100.0)PPI-clarithromycin-metronidazole-bismuth (1 [0.7])1 (100.0)PPI, proton pump inhibitor.a Dual regimen PPI-amoxicillin (1 [0.7], 100% eradication). Open table in a new tab PPI, proton pump inhibitor. Among the 92 patients who received clarithromycin-containing regimens, strains with clarithromycin resistance were statistically significantly higher among eradication-failures (13/22, 59.1%) compared with eradication successes (10/70, 14.3%; P < .01). Among the 62 patients who received metronidazole-containing regimens, strains with metronidazole resistance were statistically significantly higher among patients with eradication failures (9/18, 50.0%) than eradication successes (6/44, 13.6%; P = .002). Among the 6 patients who received metronidazole-containing regimens with eradication success and metronidazole-resistant strains, 3 regimens (50%) were quadruple therapy, and all 6 patients received ≥1 other antimicrobials to which the strain was susceptible. The rate of culture growth was 34.5% (20/58); 15 patients had antimicrobial-susceptibility data (9 clarithromycin-resistant, 6 clarithromycin-sensitive). There was no reported resistance to other antimicrobials. There was 100% concordance between clarithromycin resistance as assessed by agar dilution and NGS-based method. This report represents one of the most extensive U.S. pediatric studies on the efficacy of a NGS-based technique in characterizing microbial resistance rates in H pylori from gastric biopsies. Predictably, our cohort had the highest resistance rate to clarithromycin (23.5%) and metronidazole (21.9%). These rates are similar to the calculated U.S.-pooled prevalence and in small single-center studies in U.S. adults.6Duck W. et al.Emerg Infect Dis. 2004; 10: 1088-1094Crossref PubMed Scopus (179) Google Scholar, 7Mosites E. et al.J Glob Antimicrob Resist. 2018; 15: 148-153Crossref PubMed Scopus (21) Google Scholar, 8Argueta E.A. et al.Gastroenterology. 2021; 160: 2181-2183.e1Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Resistance rates to other antimicrobials were low as expected, because these antibiotics are infrequently used in children. Fluoroquinolone resistance rate was 8.4% compared with the 37% U.S.-pooled prevalence. Similarly, resistance to rifabutin (9, 3.6%), amoxicillin (6, 2.4%), and tetracycline (1, <1%) were low. None of the patients received rifabutin, and only one received fluoroquinolones. The overall eradication rate was 73.6%, with only quadruple therapies achieving the recommended goal for effective therapies of ≥90%. The eradication rates were lower in patients with resistant H pylori strains when compared with non-resistant. Metronidazole resistance significantly impacted eradication success. It is possible that the decreased efficacy of metronidazole in the members of our cohort with metronidazole resistance was in part due to lower dose of metronidazole or the reduced use of bismuth in metronidazole-containing regimens. NGS-based technique successfully characterized antimicrobial resistance in 92% of cases, even with the limitations of isolating DNA from paraffin blocks. The method also correlated very well with clarithromycin resistance, when compared with agar dilution. Poor culture growth due to the difficulties with isolation and cultivation of H pylori constitutes a significant problem in the United States. Only a few centers have availability of gastric biopsy culture on-site and instead rely on off-site specialty labs.4Bhakta D. et al.Clin Gastroenterol Hepatol. 2018; 16: 1531-1532Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar,5Bonilla S. et al.JPGN Reports. 2021; 2: e116Crossref Google Scholar NGS-based technique can also be performed in stool,10Moss S.F. et al.Gastroenterology. 2022; 162: 2095-2097.e2Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar further highlighting its value in pediatric population where noninvasive procedures are preferred. Limitations of our study include the regional nature of the study population, the complex population seen at tertiary care centers, which could potentially increase rates of antimicrobial resistance because of increased exposure to antibiotics (related or unrelated to H pylori), and the low rate of confirmation of eradication. In addition, the relatively small number of patients who underwent culture (the current gold standard) limits our ability to assess the validity of this assay. In conclusion, NGS-based technique provides a novel and potentially clinically useful method of characterizing resistance rates to clarithromycin and metronidazole on formalin-fixed paraffin embedded gastric biopsies. The presence of identified resistance to clarithromycin and metronidazole by NGS-based technique corresponds with lower eradication rates to common first-line regimens in children. The method is also easier and more "user friendly" than culture, although at the current time it is not reimbursable by most insurances. Although additional studies are needed to establish the correlation between NGS-based method and agar dilution in children, our preliminary data suggest the 2 tests yield similar results. The development of an easy way to ascertain antimicrobial resistance may revolutionize the treatment of H pylori by identifying a resistant population that would most benefit from quadruple therapy as initial treatment. Future studies should report on resistance and eradication rates in other U.S. regions to confirm the validity of the current clinical practices and modification of recommendations accordingly. The authors thank Samuel Zhang at American Molecular Laboratories Inc for performance of NGS antimicrobial susceptibility testing. The study was approved by the Boston Children's Hospital and Hasbro Children's Hospital IRB. Michael Herzlinger (Acquisition of data); (Review and interpretation of the data); (Revision of the manuscript) Katelyn Dannheim (Acquisition of the samples for next generation sequencing analysis); (Revision of the manuscript) Muhammad Riaz (Acquisition of the data); (Revision of the manuscript) Enju Liu (Statistical analysis and interpretation of data) Athos Bousvaros (Critical revision of the manuscript) Silvana Bonilla (Conception and design of study); (Acquisition of data); (Review and interpretation of the data); (Drafting and revision of the manuscript) PyloriAR (American Molecular Laboratories, Vernon Hills, IL) is an assay that evaluates DNA mutations or variances in the H pylori genome associated with resistance to fluoroquinolones, metronidazole, clarithromycin, amoxicillin, tetracycline, and rifabutin. We obtained five 10-mm sections from the formalin-fixed paraffin-embedded gastric biopsies collected for histopathology. Sections were sent to American Molecular Laboratories for processing. Briefly, sections were digested with lysis buffer and proteinase K, and whole DNA extracted with Qiagen spin columns and QIACube devices. A real-time polymerase chain reaction was performed to quantitatively determine H pylori presence by targeting 23S rRNA. For samples with sufficient H pylori DNA, targeted genes from the H pylori genome were amplified and enriched, and then paired-end sequencing libraries were sequenced with the IlluminaMiSeq platform (Illumina, Inc, San Diego, CA). The generated reads were mapped to the reference of H pylori 26695 genome sequence to analyze DNA mutations or variances responsible for the resistance to corresponding antimicrobials. Gastric biopsy culture specimens from Boston Children's Hospital were sent to off-site specialty lab ARUP Laboratories via courier service within the same day of biopsy procurement. Briefly, once received tissue was ground in 1 mL TSB to homogenize and subsequently inoculated on 2 Brucella agar plates. The plates are incubated at 33°C–37°C in microaerophilic conditions. Plates were examined at 3, 5, and 7 days looking for characteristic colonies. Identification was performed by matrix-assisted laser desorption ionization time-of-flight—mass spectrometry or 16S sequencing. If positive growth occurred, susceptibility testing was added. The sample then was sent by ARUP Laboratories to Mayo Laboratories (test code: ZMMLS/8073), which conducted antimicrobial susceptibilities by means of agar dilution method. The susceptibility breakpoint values for H pylori isolates used in assessing resistance to a given antibiotic are as follows (μg/mL): amoxicillin >0.25, clarithromycin >1, metronidazole >8, levofloxacin >1, and tetracycline >4.Supplementary Table 1Percentage of Antimicrobial Resistance by Type of Treatment Regimens and Eradication ResultsTreatment regimen/eradication resultNo. of strains% Resistance (n)ClaMetLevTetAmoxRifPPI-clarithromycin-amoxicillin Success5813.8 (8)22.4 (13)10.3 (6)003.4 (2) Failure2055.0 (11)20.0 (4)10.0 (2)05.0 (1)0PPI-metronidazole-amoxicillin Success19010.5 (2)0005.2 (1) Failure1020.0 (2)50.0 (5)20.0 (2)010.0 (1)0PPI-clarithromycin-metronidazole Success333.3 (1)33.3 (1)0000 Failure2100.0 (2)00000PPI-clarithromycin-amoxicillin clavulanic Success10100.0 (1)0000 Failure0N/AN/AN/AN/AN/AN/APPI-amoxicillin-doxycycline Success1000000 Failure0N/AN/AN/AN/AN/AN/APPI-amoxicillin Success1100.0 (1)00000 Failure0N/AN/AN/AN/AN/AN/APPI-metronidazole-bismuth Success0N/AN/AN/AN/AN/AN/A Failure1100 (1)100 (1)100 (1)00100 (1)PPI-amoxicillin-metronidazole-bismuth Success450.0 (2)50.0 (2)50.0 (2)025.0 (1)25.0 (1) Failure4100 (4)50.0 (2)0000PPI-tetracycline-metronidazole-bismuth Success1030.0 (3)10.0 (1)10.0 (1)0010.0 (1) Failure1100 (1)100 (1)0000PPI-doxycycline-metronidazole-bismuth Success1000000 Failure0N/AN/AN/AN/AN/AN/APPI-clarithromycin-amoxicillin-metronidazole Success520.0 (1)000020.0 (1) Failure0N/AN/AN/AN/AN/AN/APPI-clarithromycin-amoxicillin-bismuth Success1000000 Failure0N/AN/AN/AN/AN/AN/APPI-clarithromycin-metronidazole-tetracycline Success1000000 Failure0N/AN/AN/AN/AN/AN/APPI-clarithromycin-metronidazole-bismuth Success1000000 Failure0N/AN/AN/AN/AN/AN/ANOTE. Doses and frequencies of medications for all treatment regimens followed the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition 2016 revised clinical guidelines.Amox, amoxicillin; Cla, clarithromycin; Met, metronidazole; PPI, proton pump inhibitor; Rif, rifabutin; Tet, tetracycline. Open table in a new tab NOTE. Doses and frequencies of medications for all treatment regimens followed the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition 2016 revised clinical guidelines. Amox, amoxicillin; Cla, clarithromycin; Met, metronidazole; PPI, proton pump inhibitor; Rif, rifabutin; Tet, tetracycline.